Method of providing for blood count and pipette and assembly for use therein



July 24, 1962 H. w. GERARDE 3,045,494

METHOD OF PROVIDING FOR BLOOD COUNT AND PIPETTE AND ASSEMBLY FOR USETHEREIN Filed March 13, 1958 5 SheetsSheet 1 INVEN TOR. yawn-s m 524204BY H. W. GERARDE NG FOR BLOOD COUNT AND PIPE July 24, 1962 3,045,494 TTEMETHOD OF PROVIDI AND ASSEMBLY FOR USE THEREIN 3 Sheets-Sheet 2 FiledMarch 15, 1958 allillflilil II Illlnllill I! I! I INVENTOR. woe/yr: w.524295 I'M KW July 24, 1962 w GERARDE 3,045,494

METHOD OF PROVIDING FOR BLOOD COUNT AND PIPETTE AND ASSEMBLY FOR USETHEREIN Filed March 13, 1958 5 SheetsSheet 3 O as 42 44 FIG 3 INVENTOR.45 47 50 flag/4:5 W. flifl Filed Mar. 13, 1958, Ser. No. 721,139 13tCla'ims. (U. 73-4256) This invention relates to a novel method ofproviding for a blood count and also provides a structurally andfunctionally improved pipette and assembly for use in the practice ofthat method.

This application is a continuation-in-part of my prior United Statesapplication for patent entitled Disposable Pipette Assembly, bearingSerial No. 642,583 and filed on February 26, 1957, now abandoned.

It is a primary object of the invention to furnish a pipette andassembly which, because of their improved functional accuracy, low cost,and simplicity and efiiciency of operation, will encourage the greateruse of blood counting and similar laboratory techniques for diagnosticpurposes.

By means of the present teachings a structure is furnished which may beproduced at nominal cost. Therefore the assembly may have a one-timeuse, after which it is discarded. Accordingly, the danger of infectionto the user, as existing with the use of conventional pipettes andassemblies, is avoided. This is in addition to the fact that thestructure will not require cleaning after use and may be employed bytechnicians who need not be as highly skilled and trained as hasheretofore been required where conventional pipettes and assemblies havebeen used.

With these and other objects in mind, reference is had to the attachedsheets of drawings illustrating practical embodiments of the inventionand in which:

FIG. 1 is a perspective view of a pipette assembly;

FIG. 2 is a partly sectional and fragmentary view of the assembly asshown in FIG. 1;

FIG. 3 is a fragmentary sectionalview showing an alternative structurewhich may be employed;

FIGS. 4 to 9 inclusive are somewhat schematic and partly sectional viewsillustrative of the steps followed in utilizing one form of pipetteassembly embracing the present teachings;

FIG. 10 is a fragmentary sectional side view of an alternative form ofassembly and showing the same with the parts in one position; a

FIG. 11 is a similar view, but showing the parts in a differentposition;

FIG. 12 is a partly sectional and fragmentary view illustrative of afurther form of assembly; and

FIG. 13 is a similar view of still another form. of such assembly.

Referring primarily to FIGS. 1 and 2, in which a preferred form ofpipette assembly has been shown, the numeral 15 indicates the capillarytube, the inner end of which is mounted by a sleeve 16, in turnconnected to a reservoir and actuating unit 17. Tube 15 provides thepipette proper. While it may be produced by employing a proper plasticmaterial of the resin type, it is preferred that it be manufactured ofglass. Sleeve 16 may be formed of a resin, although other materialscould be employed. Reservoir 17 should be compressible to vary itsinternal volume. It should be formed of a plastic such astrifluorchloroethylene or vinydene chloride co-poly'mer, both of whichwill hold diluent for long periods of time without loss. Also, both maybe used in sufficiently thin gauge so that the reservoir body willembrace flexible and resilient characteristics.

The forward end of sleeve 16 preferably defines a tip States ate-tfiidgd Patented .iuly 24-, 1&62

, secured in this position in any desired manner and has its adjacentend terminating in the zone of the collar and Wall. A sheath 20, asindicated in dotted lines in FIG.- 1, may enclose tube 15 to protect thelatter and bear against the surface of tip portion 18, from which it mayreadily be separated when the assembly is to be used.

The rear end of sleeve 16 terminates in a reduced flange 21 which isreceived within the boreof collar 22 formed at the forward end ofreservoir 17. A manipulating member or part is conveniently mounted bysleeve 16 and may take the form of an integral tab 23 to which a label23 may be suitably attached. This label presents a surface to receiveproper indicia, which may include, for example, the date, specimennumber, the patients name, etc. A volume of diluent 2d of a typecorresponding to the count to be performed and proportioned to thevolume of pipette tube 15 is disposed within reservoir 17. Alsoextending into the reservoir interior is the head portion 25 of a mixingbead provided with a plug or stem 26.

That stern has a diameter such that it extends into the bore of sleeve16 to seal the latter. The area of head 25 is preferably greater thanthe bore defined by collar 22. Therefore, with the parts in thepositions shown in FIGS. 1 and 2, the diluent body 24 within thereservoir will not pass into the bore of sleeve 16 or other parts of theassembly. Should the sleeve be detached from the reservoir 17, then thebead will be retained by the latter and will drop into its interior uponthe reservoir being shifted to an upright position. i

If it were desired to form the reservoir of glass, this of course couldreadily be done by employing a structure of the general character shownin FIG. 3. In that view the numeral 27 indicates the wall of such aglass reservoir. In common with reservoir 17, it will contain a body ofdiluent 28. In order to provide for conditions of pressure above andbelow atmospheric, body 27 may at its rear end define a neck 28. To thelatter there is applied a bulb 29 conveniently formed of rubber. It isapparent that by compressing this bulb and then allowing it to expand,an effect is accomplished corresponding to that achieved by compressingreservoir 17 and then allowing it to expand. If a construction such asis illustrated in FIG. 3 is employed, then, of course, the forward endof the reservoir 27 may have attached to it a sleeve such as 16, formedof plastic or other elected material, and in turn carrying a label ortag 23 of, for example, paper, and mounting a capillary pipette tube,preferably of glass.

Such a tube will have a bore surface which is completely clean. The thinwall of the pipette is provided by drawing a clean tube of larger borein a flame. When reduced to proper size, the tube is cut by cleavage,after the tube surface has been scratched with a diamond or Carborundumwheel. In this manner the outer end surface of the pipette is perfectlyclean, involving as it does cleavage of crystalline material. Theoutside diameter of tube 15 should be less than the area of a drop ofblood into which its outer end is to be dipped or with which that end isto be placed in contact. Therefore, the tube may involve an outsidediameter of .036" with a plus or minus factor on the order of .001". Thebore diameter, according to the present teaching, should preferably beon the order of .0285" with a plus or minus factor of .0004. 1

This will provide a pipette involving a capillary tube in which thecapacity of the latter is in direct proportion to its length. An ideallength, based on the foregoing measurements, would be one in which .013cc. with a plus or minus factor of .0002 cc. is provided for. Thediluent within the reservoir or container might vary, of course,according to the type of liquid which is to receive analysis. However,in the case of blood, for which the present apparatus is primarilydesigned, the volume of diluent, in connection with erythrocyte (redcell) count would involve 2.609 cc. with a plus or minus variation of.031 cc., while in the case of leukocyte (white cell) count the volumeof diluent would be .2609 cc. with a plus or minus factor on the orderof .0031 cc. The diluent solutions contemplated would in the case of'redcells, preferably involve:

Distilled water ml 200.0 Sodium chloride gm 1.0 Sodium sulfate gm 5.0Mercuric chloride gm 0.5

In connection with the determination of a white cell count, 1 percent ofhydrochloric acid in water will ordinarily be employed.

While, as afore brought out, in assemblies employed respectively for redand white cell counts th capacity of the capillary tubes might beidentical while the volume of diluent in the reservoirs of thoseassemblies were different, the reverse might be true. In other words,for a red cell count the accepted proportion of blood to diluent is 1part of the former of 200 parts of the latter. In the case of a whitecell count, 1 part of blood is employed to 200 parts of diluent. -Underthese circumstances it is feasible to employ capillary tubes differingin volume by either length and/ or bore diameter. In the case of a redcell count, the volume of the capillary tube could conveniently be .013cc., while in the case of a white cell count the volume of the capillarytube could be .0013 cc.

Where the capillary tube embodies glass, it has been found that it is solight that even if dropped on a relatively hard surface, it will notshatter. Whether it be made of glass or of a suitable plastic,ordinarily its outside diameter should not substantially exceed 1 mm.Where the volumes of the capillary tubes are different for red and whitecell counts, then, of course, the same size reservoirs may be used inthe different assemblies. Likewise, the same volume of diluent may becontained in each reservoir.

According to the present concept, the blood will rise in the capillarytube to completely fill the bore of the latter. The rate of penetration,penetrating power or penerating pressure of liquids such as blood intocapillary tubes is dependent on the surface tensiond and the advancingcontact angle of the liquid. Any increase in the contact angle is to beavoided. With the bore surface of the capillary tube being completelyclean, no film on that surface will be formed. It has been found thatfilms which are only one molecule thick may profoundly alter theproperties of the bore surface. By providing a tube the outer end ofwhich is defined by a thin wall, a sharp edge is present which reducesor eliminates the contact angle, with resultant increased adhesiontension or wetting power. The surface of this edge, being formed bycleavage of the tube, has a crystalline structure that is different fromthe outer and inner wall surfaces of that tube. This edge surfaceapproaches being the cleanest and most perfect surface attainable bycleavage of a single crystal. The contact angle of a liquid such aswater with this surface is as close to zero as can be. Thus, while it isfeasible in assemblies as herein taught to provid capillary tubes ofmaterials other than glass, it is definitely preferred to utilize glasstubing produced in the manner afore outlined.

Assuming that an apparatus of the type shown in FIGS. 1 and 2 is to beemployed, the physician or technician will, for example, lance the tipof the patients finger, so that (FIG. 4) a drop 30 of blood appears onthe surface of that finger. Reservoir 17 will be held in at least anupwardly inclined position and sleeve 16 detached therefrom. In suchdetachment the bead will, of course, drop into the interior of thereservoir. The sleeve, together with the pipette tube 15 mountedthereby, may now be grasped, or if a tab 23 be associated therewith, thelatter may be gripped to furnish a manipulating unit. In any event, bymaintaining tube 15 at an angle slightly inclined upwardly from thehorizontal, its outer end may be brought into contact with the surfaceof drop 30. As indicated in FIG. 4A, the contact angl of the blood withthe bore surface 31 of the tube 15 will approach zero. Also, the endarea of the tube will be less than the area of the blood drop. The bloodtherefore rises in the tube to fill the latter completely by capillaryattraction, because the adhesive force between the glass surface of thisbore and the blood is greater than the cohesive force of the bloodmolecules themselves. Accordingly, the column of blood will extendthroughout the entire length of the bore 3-1 and terminate at its upperor inner end in line with the edge of tube 15. This column will remainintact under all circumstances, so that it is unnecessary to resort toprocedures as heretofore practiced to prevent a portion of the columnbeing accidental-1y displaced from the bore.

Therefore, as shown in FIG. 5, the outer end of the capillary tube maybe introduced into the reservoir 17. At the time of that introductionthe walls of the reservoir will be partially compressed, as alsoillustrated in FIG. 5. As sleeve 16 is brought into sealing engagementwith the bore surface of collar 22, the pressure upon the side walls isreleased, as illustrated in FIG. 6. Accordingly, an aspirating actionoccurs which serves to draw the entire liquid column within tube 15 intothe body of diluent within the reservoir. To scavenge substantially alltraces of blood from the bore surface, the reservoir may now again becompressed, as in FIG. 7. This will force diluent up through the bore ofthe tube and into the sleeve 16. With the release of this compressiveaction, the displaced liquid is again drawn into the reservoir.

Now, as shown in FIG. 8, the sleeve is unseated from the reservoir andreversed, so that tube 15 extends outwardly. The reservoir is shaken, sothat the mixing bead will be agitated within the solution contained inthe reservoir. This will assure an even dispersion of blood throughoutthe body of the solution. When this has been achieved, and as shown inFIG. 9, a desired volume of the solution may be discharged, for example,upon a counting chamber 32, to which a glass 33 had been applied inaccordance with conventional techniques.

Thus, in theassembly as described, the pipette tube 15 collects andmeasures blood accurately in a single automatic operation, which isinstantly initiated simply by touching the tip of the capillary to thedrop of blood. The assembly collects and measures the blood, since thelatter is drawn into the tube bore by capillary action and is measuredby the length of the tube. Accordingly, unerring physical forces andfactors are substituted for the human judgment, skill and experiencerequired to carry out the same operation with conventional equipment.The blood and diluent are not drawn into the tube by mouth. The requiredvolumes are definitely achieved, rather than by relying on thetechnician attempting to line up the meniscus at the top of a liquidcolumn with a mark on the outside of the tube. Therefore, there iseliminated one of the principal sources of human error inherent incollecting and measuring blood and diluent with the equipment generallyused for the purpose at the present time. The blood is collected rapidlyand accurately by a quickly performed operation involving the drawing ofthe blood out of the capillary tube into the reservoir. The lattercontains a precisely predetermined and measured volume of diluent. Alloperations are carried out with the use of the hands and without thenecessity of visually determining proper volumes requiring individualcalibration of the tube.

With conventional pipette techniques, it is extremely difiicult tocontrol the rate of flow and size of the drop forming at the tip.Employing the present thin-walled capillary tube, the operator ortechnician has precise, positive control of the flow of diluted bloodfrom the reservoir to the tip of the capillary, as illustrated in FIG.9. Diluted blood is forced out of the reservoir by simply applyingpressure to the walls of the latter, rather than by allowing thesolution to flow out under the influence of gravity. It is apparent thatif a construction such as that illustrated in FIG. 3 is employed in lieuof that shown in FIGS. 1 and 2, the same desirable results are achieved.This is also true of the structures shown in FIGS. to 13 inclusive.

In connection with FIG. 12, the numeral 34 indicates a reservoircorresponding to reservoir 17 and containing a predetermined volume ofdiluent 35. Within this reservoir a mixing bead 36 of suitableconfiguration is disposed. The forward end of the reservoir is definedby a wall through which a passage 37 extends. That passage is initiallyclosed by a stopper portion 38 formed in the base portion of an adaptorand mounting member involvinga cylindrical body 39 preferably formedwith an outstanding fiange 4th at the end opposite reservoir 34. Thisadaptor serves to mount the base portion of a lancet 4 1, the point ofwhich extends beyond the adaptor. Also, it serves to support a sleeve 42corresponding to sleeve 16 and also supporting the inner end of acapillary tube 43 corresponding to tube 15.

Thus, the physician or technician is provided in the assembly of FIG. 12with an instrument such that the drop of blood may be created withoutresorting to an element foreign to the assembly. The user will simplydismount the adaptor 39 from the reservoir 3%, while maintaining thelatter in an upright position so that diluent does not flow outwardlythrough the bore 37. Now, by detaching sleeve 42 from the adaptor, thepiercing point of the lancet is available for pricking the finger orother epidermis surface of the patient. To this end the adaptor itselfmay serve as a manipulating element for projecting and retracting thesterile lancer point with respect to the surface tobe pierced. Theadaptor may now be discarded. Thereupon, the technique as aforetraversed maybe followed in detail in order to provide the dilutedsolution for blood count purposes.

In certain instances it may be desired to furnish an assembly in whichthe diluent will be sealed against contamination or escape except whenparts of the assembly are destroyed. Such a design is illustrated inFIG. 13, in which the numeral 44 indicates the reservoir, whichcorresponds to reservoir 17 and contains a body of diluent 45. Also,this reservoir preferably contains a mixing bead or element 47. Theforward end wall 48 of the reservoir maintains the interior of the sameisolated. The outer face of end wall 48 is provided with a recess thebase of which defines a. diaphragm portion 459. This recess has adiameter such that the rear end of a sleeve 50, corresponding to sleeve16, will be accommmodate-d' and retained therein by frictional contactof the surfaces. The sleeve in turn supports a capillary tube 51corresponding to tube 15.

When a device such as this is to be used, then the operator will simplygrip sleeve 50 and force the same toward the reservoir. The edge of thatsleeve will be sufficiently sharp to assure a rupturing of diaphragmportion 49. It will of course be understood that the material of thesleeve 50 should also be sufficiently hard to assure the desiredpenetration. When this result has been obtained, then the procedure asheretofore outlined will be followed.

Finally, considering the device as illustrated in FIGS. 10 and 11, thenumeral 52 indicates a reservoir preferably formed of plastic materialso as to be compressible. The body of the reservoir is closed at one endby a wall 53 having a bore or opening, while at the other end it isclosed by a wall provided with an opening which may be defined by atubular tip 54. The reservoir contains a body of diluent 55 related tothe volume of blood or other liquid provided by the bore of thecapillary tube forming a part of the assembly. A mixing bead 56 may bedisposed within the reservoir 52. An actuator, preferably in the form ofa tube of plastic material, as indicated at 57, extends through theopening in wall 53. At its inner end it serves to mount the adjacent endof a capillary tube 58 corresponding to the tube 15.

The device will come to the physician or technician in the conditionshown in FIG. 10. Tube 58 may be housed by a sheath (not shown).Likewise, tube 57 may be suitably protected or sealed. The finger of thepatient will be lanced, and the outer end of capillary tube 58 will bebrought into engagement with the resulting drop of blood by incliningtube 58 in an upward direction, as illustrated in FIG. 4A. Under thesecircumstances the bore of tube 58 will automatically draw a volume ofblood upwardly by capillary action until the bore of that tube isfilled.

Thus, a predetermined volume of blood is collected which is the desiredquantity when related to the volume of diluent 55. By virtue of thesleeve furnished by the inner end of tube 57, the column of blood willnot pass beyond the inner end of tube 58. Using tube 57 as amanipulating element, tube 58 is retracted to a point where it is whollycontained within reservoir 52, as shown in FIG. 11.

The walls of that reservoir are. now compressed, as indicated in dottedand dash lines in the latter figure. The bore defined by tip 54 issealed by, for example, placing the operators finger in contact with theedge of that tip. Now, upon releasing the pressure upon the walls ofreservoir 52, an aspirating action occurs such that the column of bloodwithin tube 58 is drawn into the interior of the reservoir. If desired,a scavening action may be effected by simply maintaining the seal on tip54, bringing the reservoir to an upright position where the tube 57extends upwardly therefrom, and thereupon compressing and releasingreservoir 52 a number of times. Under these-circumstances the solutionwill pass into and even beyond the bore of tube 58 and then be loweredto where the column is entirely discharged within the interior of thereservoir.

Now, by agitating the assembly, head 56 assures an even dispersion ofthe blood throughout the solution. By inclining the reservoir so thattip 54 extends downwardly, a pressure on its walls will. serve todispense desired amounts of solution onto a receiving surface such as acounting chamber.

In common with the other devices and portions of the assemblies as shownin preceding views, the entire apparatus may be discarded after a singleuse. No cleaning of the parts will therefore be necessary after a finalamount of solution has been dispensed. While the present apparatus isintended primarily for use in connection with blood counts, it isapparent that it will also: be useful in connection with analytical Workinvolving liquids other than blood.

Thus, among others, the several objects of the invention as specificallyaforenoted are achieved. Qbviously, numerous changes in construction maybe resorted to and the steps of the method may be varied withoutdeparting from the spirit of the invention as defined by the claims.

, I claim:

' 1. A pipette assembly including in combination a flexible reservoircontaining a predetermined amount of diluent and having one open end, acapillary tube having a blood-receiving bore capacity precisely relatedto the amount of diluent, means for detachably connecting one end ofsaid tube with the open reservoir end, said connecting means including asleeve having one end permanently connected to said tube, said sleevehaving a bore of greater diameter than the tube bore and incommunication with the latter, the sleeve adjacent its opposite endproviding a surface to seaiingly and detachably engage with a surface ofsaid reservoir, and means for initially sealing the bore of said sleeveagainst a flow of liquid from the reservoir into said sleeve.

2. In an assembly as defined in claim 1, said sealing means comprisingan agitating element having a body disposed within said reservoir and ofa size larger than the open end thereof and a stem extending throughsuch end and in detachable engagement with the bore surface of saidsleeve.

3. A pipette assembly including in combination a capillary tube havingan outer end, such end being contaotable with epidermis supporting adrop of blood to immerse that end in the blood, the inner tube end beingopen to the atmosphere, whereby blood will flow solely by capillaryaction through the tube to entirely fill the bore of the latter and bemaintained by capillary attraction therein as said tube is subsequentlyremoved from a position adjacent the epidermis, a separate reservoircontaining a volume of diluent precisely related to the capacity of thetube bore, said reservoir having an opening, the outer end of said tubebeing insertable through said opening to extend into the interior ofsaid reservoir for the discharge of blood theretofore retained withinthe tube bore, and means providing a mounting and seal between the edgesof said opening and tube to detachably retain the latter in suchposition.

4. In an assembly as defined in claim 3, means for causing a fiow of airthrough the thus retained tube from its inner to its outer end wherebyto discharge the blood within the tube bore into said reservoir.

5. In an assembly as defined in claim 4, at least a part of saidreservoir being flexible and resilient to diminish the interior capacitythereof when subjected to compression and to increase such capacity whenreturning to a normal position, such return resulting in said air flow.

6. In an assembly as defined in claim 3, said mounting and sealcomprising a sleeve having a bore larger than the diameter of said tubeand secured to said tube adjacent its inner end.

7. In an assembly as defined in claim 6, a tube-manipulating partextending outwardly from said sleeve and furnishing indicia-receivingsurfaces.

8. In an assembly as defined in claim 6, an end wall forming a part ofsaid sleeve and the inner end of such tube extending to a pointsubstantially in line with said wall.

9. In an assembly as defined in claim 3, a plug initially obstructingcommunication between the outer atmosphere and the interior of saidreservoir and said plug being displaceable into the reservoir interiorto provide a mixing bead therein.

10. In an assembly as defined in claim 6, said sleeve providing aliquid-receiving part in communication with the tube bore and extendingoutwardly of said reservoir with said tube inserted in the latter andthe resilient part of said reservoir being thereupon compressible toforce liquid outwardly through the tube bore into said receiving part.

11. In an assembly as defined in claim 8, said sleeve bearing againstsuch opening edges, a plug initially closing the bore of said sleeve andthe head of said plug having an area larger than that of said openingand extending into the interior of said reservoir to drop Within thelatter and function as a mixing bead as said sleeve and tube aredetached from said reservoir.

12. For use with a pipette assembly involving a separate capillary tubeto be detachably mounted thereby, a reservoir to receive diluent andformed with an opening the edge zones of which define surfaces forengagement with sunfaces ancillary to said tube to mount the latter, aplug obstructing said opening and said plug being displaceable into thereservoir interior to provide a mixing head for the liquid therein.

13. In a structure as defined in claim 12, a head portion forming a partof said plug, said portion having an area greater than that of saidopening and lying within the reservoir interior whereby said plug maynot be separated from said reservoir.

References Cited in the file of this patent UNITED STATES PATENTS766,204 Walsh Aug. 2, 1904 1,482,966 Bevan Feb. 5, 1924 1,594,370 KubotaAug. 3, 1926 2,540,364 Adams Feb. 6, 1951 2,599,446 Greene June 3, 19522,693,183 Loclchart Nov. 2, 1954 2,737,812 Haak Mar. 13, 1956 OTHERREFERENCES Quantitative Ultra Micro Analysis, by Paul L. Kirk, publishedby John Wiley & Sons, New York, 1950 (pages 22 and 23 pertinent). (Copyin Scientific Library.)

